Extra column band broadening in detectors has been paid attention to since high performance liquid chromatography (HPLC) was introduced. Accordingly, as columns and injectors have been improved the requirement on detector performance has increased to minimize peak broadening by dispersion. In this respect the cell volume as well as connections play an important role. The whole detector arrangement may influence the flow properties which in turn affect the band spreading.
There is a growing interest in the use of miniaturized HPLC systems based on packed microbore columns or open tubular columns. These chromatographic systems may exhibit high resolving power together with a low flow rate of the mobile phase. The microbore packed column technique was introduced by Scott and Kucera [1,2] (for these and other references indicated in brackets please refer to list at end of the specification). They pointed out the importance of diminishing the detector cell volume to utilize the separation effiency of the column. Ishii et al [3,4] constructed a miniaturized HPLC system with open tubular columns which have essentially smaller inner diameters of about 50 .mu.m. With this technique the detector cell requires still smaller volumes, in the range of 0.1-1 .mu.l. Knox and Gilbert [5] calculated that the effective detection volumes should be in the order of 1-10 nl before there is any hope of operating capillary HPLC systems under optimal conditions. They also stated that the practical limitation to capillary HPLC arises from the dispersion by the detector.
When considering different small volume detectors the fluorescence technique is promising because of its high sensitivity. HPLC fluorescence detectors utilizing laser excitation radiation have recently been introduced [6, 7, 8]. Laser fluorimetry has some important characteristics which may be advantageous with small volume detectors. The produced emission radiation is directly proportional to the intense laser excitation light which leads to extreme sensitivity. Another property is the spatial coherence of the laser beam which facilitates the irradiation of small detector volumes. In addition the monochromaticity of the laser makes it easier to suppress scattered light from Rayleigh and Raman processes as well as reflexions without sacrificing sensitivity.
Diebold and Zare [6] presented a windowless flow cell where the HPLC effluents flow from a steel capillary tube down to a rod 2 mm below forming a droplet bridge of 4 .mu.l volume. Focusing the laser beam to a small spot inside the droplet facilitates rejecting the elastically scattered light. With a pulsed HeCd laser and the use of gated detection electronics they achieved very high sensitivity when determining aflatoxins. Hershberger et al. [7] designed a HPLC cell based on the sheath flow principle where the affluent is injected in the center of an ensheathing solvent stream under laminar flow conditions. The laser beam enlighted cell volume is very small or in the range of 6-150 nl. Since the windows are not in contact with the sample flow the stray light from the windows is reduced. Sepaniak and Yeung [8] used a quartz capillary tube where the HPLC effluent moved upwards. A focused laser beam was placed in the effluent underneath an optical fiber. The emitted light was collected through the fiber perpendicular to the laser beam. The construction leads to a minimal influence of scattered and fluorescent light from the capillary tube walls. The limiting factors of fluorescence detectability are to a great part connected with background noise from the cell, optical components, solvent and sample contaminants. The reduction of the background emission is highly dependent on the detector cell design. The objective of this invention is to design a laser based detector ideal to use with conventional HPLC columns as well as microbore columns. Special attention has been paid to the effective cell volume regarding extra column effects by comparing the efficiency of the two systems.